miR-16 and miR-103 impact 5-HT4 receptor signalling and correlate with symptom profile in irritable bowel syndrome

Abstract

Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT₄ receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HT₄R selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.*61 T > C within the 5-HT₄ receptor gene HTR4 to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms HTR4b/i and putatively impairs HTR4 expression. Subsequent miRNA-profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. In vitro assays confirmed expression regulation via three 3'UTR binding sites. The novel isoform HTR4b_2 lacking two of the three miRNA binding sites escapes miR-16/103/107 regulation in SNP carriers. We provide the first evidence that HTR4 expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.*61 T > C or by diminished levels of miR-16 and miR-103 suggesting that HTR4 might be involved in the development of IBS-D.

Figure 1

Illustration of HTR4 isoforms and their expression pattern in the human GI tract. (A) HTR4 isoforms HTR4a, HTR4b, HTR4d, HTR4g, HTR4i and HTR4c, without a specified 3′UTR. The common region (dark grey) encompasses exons 2-6 and is shared by all six HTR4 isoforms. The isoform-specific 3′UTRs are highlighted in red and encode unique 5-HT₄R C-termini. Not drawn to scale. (A′) 5-HT₄ receptor isoform transmembrane topologies indicating individual C-terminal ends predicted by Protter for visualization of proteoforms (http://wlab.ethz.ch/protter). (B) Expression pattern of HTR4 isoforms in different human GI regions. ARF1 served as mRNA integrity and loading control. Respective PCR images were cropped for figure implementation.

Figure 2

Schematic illustration of the miR-16 family and miR-103/miR-107 binding sites in the 3′UTR of the HTR4b/i isoforms. Binding sites (I-III) are based on predictions by TargetScan, miRanda and/or RegRNA. The position of HTR4b/i c.*61 T > C is highlighted in red within the seed region (nucleotides 2–8) of the indicated miRNA binding site.

Figure 3

Identification of two novel HTR4b splice variants by 3′RACE. (A) Three different isoforms were identified by 3′RACE. (A′) Schematic illustration of the 3′UTRs of full length HTR4b and the novel isoforms HTR4b_2 and HTR4b_3. miRNA binding sites are indicated by ‘I-III’ (in blue). Arrows reflect positions of 3′RACE primers. (B) Expression pattern of the three HTR4b isoforms in different human GI tissues. ARF1 served as cDNA integrity and loading control. Respective PCR images were cropped for figure implementation. (C) Relative luciferase activity of HTR4b_2 and HTR4b 3′UTR reporter gene constructs (n = 3) and (D) Relative 5-HT_4b and _b2 receptor levels quantified by In Cell Western experiments (n = 3) in HEK293T. Values are means ± SEM., **p < 0.01, ***p < 0.001. Unpaired t-test.

Figure 4

Expression analyses of relevant miRNAs, HTR4b and HTR4b_2 in human colonic subregions. nCounter miRNA expression profile of (A) selected miR-16 family members as well as of miR-103/miR-107. (B) HTR4b and (C) HTR4b_2 in normal laser capture microdissected human colonic subregions. Values are means ± SEM of codeset counts from total RNA of tissue specimens from four individuals, respectively. E (epithelium), LP (lamina propria), M (muscle), MP (myenteric plexus).

Figure 5

HTR4b mRNA levels after overexpression of several miRNAs in Colo320 cells analysed by qPCR. Relative expression analysis of HTR4b mRNA levels after transfection (72 h) with different miRNAs (miR-15b, miR-16, miR-497, miR-15b/16/497, miR-103) and a negative control miR (neg. ctrl. miR). Values are means ± SEM of three to four independent experiments and were normalised to SDHA. *p < 0.05; ***p < 0.001. Unpaired t-test.

Figure 6

The HTR4b_2 novel isoform carrying the c.*61 T > C SNP escapes miRNA regulation. Illustration of the HTR4b/b_2 related luciferase reporter gene expression experiments. (A) Respective HTR4b/i 3′UTRs including predicted miRNA target sites (I-III, indicated in blue) and luciferase gene reporter constructs. (A′) Relative luciferase activity in HEK293T transfected with miR-16 family members. (A″) Relative luciferase activity in Colo320 transfected with miR-103. (A′) and (A″) Particular miRNAs are both co-expressed with respective luciferase constructs (WT, c.*61 C, mut2, mut3, mut4) and related to negative control miRNA (neg. ctrl. miR). Values are means ± SEM. n = 4 (A′) and n = 3 (A″) experiments for each condition. (B) Respective HTR4b_2 3′UTR including predicted miRNA target site (I) and luciferase gene reporter constructs. (B′) Relative luciferase activity in Colo320 transfected with miR-15b/16/497 (miR-16 family) or (B″) relative luciferase activity in Colo320 transfected with miR-103. (B′) and (B″) Particular miRNAs are both co-expressed with luciferase constructs (WT, c.*61 C, mut1) or negative control miRNA (neg. ctrl. miR). Values are means ± SEM. (n = 5) (B′) and (n = 4) (B″) experiments for each condition. *p < 0.05; **p < 0.01; ***p < 0.001. One-way ANOVA with Bonferroni post-hoc test, WT (wild type), mut (mutated).

Figure 7

Expression analysis of miR-16 and miR-103 and HTR4b and HTR4b_2 in the human jejunal mucosa in IBS-D vs. healthy controls (Ctrl.). (A) qPCR analysis of miR-16 and miR-103. Fold-change value is based on the ratio of target miRNA and the average of reference genes normalised to the average of the healthy group. Values are means ± SEM. (14 IBS-D; 17 controls). **p < 0.01. Unpaired t-test with Welch’s correction. (B) nCounter analyses of HTR4b and HTR4b_2. Fold-change is based on the ratio between target mRNA and the average of the reference genes normalised to the average of the healthy control group. Values are means ± SEM. (30 IBS-D; 18 controls). Mann-Whitney U test. (C) Correlations of IBS symptoms with miRNA expression in the jejunal mucosa of IBS-D patients and controls. Spearman’s correlation rho was applied to the pooled data. Identical values refer to multiple equal correlation values of different individuals. n = 31 (14 IBS-D; 17 controls). Spearman’s rho (r_s) and p-values (p) are given.

Figure 8

5-HT₄ receptor mediated function in the intestine. 5-HT₄ receptor activation promotes GI motility at different levels. Decreased miR-16 and miR-103 levels as well as hypermorphic allele variants (c.*61 T > C) may lead to elevated 5-HT₄ receptor activity resulting in increased secretion and peristalsis in all or particular subregions and make individuals more susceptible to develop diarrhoea. Figure components were kindly provided from Servier Medical Art (http://www.servier.com). This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA. The authors acknowledge the free figure access.